Lytic Activity Of Phages Research Articles

An integrated three-signal biosensor based on phage multifunctional probe for simultaneous and ultrasensitive detection of live /dead Listeria monocytogenes

The simultaneous and sensitive detection of live/dead bacteria in food samples is crucial for the control and prevention of foodborne pathogen contamination. Herein, a novel three-signal biosensor for simultaneous and ultrasensitive detection of live/dead Listeria monocytogenes (L.m) was developed. Concretely, a phage/prussian blue nanoparticles (PBNPs) and phage/polyethyleneimine@magnetic nanoparticles (PEI@MNPs) multifunctional probes were prepared. In the presence of L.m, a sandwich complex of phage/PBNPs-L.m-phage/PEI@MNPs was formed and separated by magnetic field. With the lytic activity of phages, the bioluminescent signal generated by adenosine triphosphate (ATP) was utilized for the quantification of live L.m. Photothermal and colorimetric signals triggered by the PBNPs could quantitative determination of total L.m. The combined results of total/live bacteria detection facilitate the calculation of dead bacteria. Bioluminescent signal allows for the precise detection of live L.m with a limit of detection (LOD) of 1 CFU/mL, while the photothermal and colorimetric signals enable on-site measurement of total bacteria with a LOD of 5 CFU/mL. This biosensor exhibits the multiple signals, realizing the diversification of bacterial information output. Meanwhile, the three signals can realize the mutually support, thereby improving the accuracy of detection results. This work opens a new avenue for the simple, ultrasensitive and simultaneous quantification of both live and dead bacteria.

Isolation and Characterization of Lytic Bacteriophages Specific for Campylobacter jejuni and Campylobacter coli

In this study, two lytic bacteriophages designated as vB_CjP and vB_CcM were isolated and evaluated for their ability to combat multidrug-resistant bacteria Campylobacter jejuni and Campylobacter coli, respectively. A morphological analysis of these phages by transmission electron microscopy revealed that the vB-CjP bacteriophage had a mean head dimension of 66.6 ± 2.1 nm and a short non-contractile tail and belongs to the Podoviridae family, whereas vB_CcM had a mean head dimension of 80 ± 3.2 nm, a contractile tail, and a length calculated to be 60 ± 2.5 nm and belongs to the Myoviridae family. The results of the host range assay showed that vB_CjP could infect 5 of 10 C. jejuni isolates, whereas vB_CcM could infect 4 of 10 C. coli isolates. Both phages were thermostable and did not lose their infectivity and ability to lyse their host following exposure to 60 °C for 10 min; furthermore, phage particles were relatively stable within a pH range of 6–8. A one-step growth curve indicated that the phages produced estimated burst sizes of 110 and 120 PFU per infected cell with latent periods of 10 and 15 min, for vB-CjP and vB-CcM, respectively. The lytic activity of these phages against planktonic Campylobacter showed that these phages were able to control the growth of Campylobacter in vitro. These results suggest that these phages have a high potential for phage applications and can reduce significantly the counts of Campylobacter spp. The lytic activity of vB-CjP and vB-CcM phages at different (MOIs) against multidrug resistance Campylobacter strains was evaluated. The bacterial growth was slightly delayed by both phages, and the highest efficiency of both phages was observed when MOI = 1 was applied.

Application of phage cocktail for control of shiga toxin-producing Escherichia coli in foods and food contact surfaces

Background: Shiga toxin-producing Escherichia coli; STEC remains a global public health and food safety challenge. Despite the various control measures available, there are still reports of outbreak and illness caused by this pathogen. Therefore, it is important to reduce this pathogen along the food chain. Methods and materials: In this study, seven STEC phages were isolated from irrigation ponds and streams. The host range and lytic activity of phages were characterized using 120 STEC strains previously isolated from foods. CFM1, WWM1 and WWM6 showed a wide host range and broad lytic activity. A phage cocktail designated CCD1 containing the three lytic phages for STEC was tested for its ability to reduce contamination of hard surfaces (gypsum board), fresh vegetables (lettuce, cabbage, cucumber and carrot). The samples were experimentally contaminated using a mixture of five STEC strains and were treated with CCD1 (test sample) and sterile SM buffer (control samples) at 4 °C and 25 °C. Results: After 10 minutes of treatment with the CCD1 cocktail using different phage concentration (1010, 109, and 108 PFU/ml), statistically significant reductions (P = < 0.05) of 99.50%, 97%, and 95%, respectively were observed in the number of viable STEC recovered from the gypsum board surface. Also, reduction was observed in the fresh vegetables samples ranging from 95% (at 30 mins) to 99.99% (at 12 hours). Conclusion: Findings from this study suggest that phages can be used to decontaminate vegetables and food contact surfaces. Its application as biocontrol could be useful in the prevention of food borne illnesses and outbreaks.

The antibacterial effect of chitosan‐based edible coating incorporated with a lytic bacteriophage against Escherichia coli O157:H7 on the surface of tomatoes

AbstractEdible coating can be used as carriers of bacteriophages, whose release into the food surface could control bacterial growth. The aim of the study was to isolate and characterize bacteriophages against a wide range of E. coli strains. Furthermore, the efficacy of chitosan‐based edible coating incorporated with a lytic bacteriophage in the biological control of E. coli on the surface of tomatoes was evaluated and discussed. Three phages were isolated from raw beef and goat stool samples, and were characterized by host range, virion structure, and DNA restriction profiles. Analysis by electron microscopy revealed that the three phages belong to the Caudovirales order. Based on the host range of the phages, they have wide infection abilities against the Enterobacteriaceae family. Phages isolated have dsDNA genomes at about 40 kbp according to restriction digest of complete phage genome. The results demonstrate that the chitosan‐based edible coating can stabilize phage vB_EcoMH2W without significant loss in lytic activity of phage over a period of one week. Moreover, the results of bacterial growth analysis showed an approximately 3 log differences in microbial levels between the control and the treatment samples. Therefore, chitosan as edible coating would enhance the microbial safety of foods during storage.Practical applicationsThe development of functional edible coatings formulations incorporated with antimicrobial agents to minimize the risk of foodborne contamination by pathogens, inactivate pathogens present in the food, and limit pathogen growth is an environmentally friendly alternative that offers substantial advantages. Several antimicrobial agents, including phages, that can be incorporated into edible coating as packaging strategy to reducing levels of pathogenic bacterial. The edible coating incorporated with lytic phages have several potential applications in the food industry as a natural method for biocontrol of food‐borne pathogens, which can be released into the food slowly and steadily extending the antimicrobial activity.

Using Phage PSM‐1 to Control Shewanella marisflavi Infection in Juvenile Sea Cucumber, Apostichopus japonicus

AbstractShewanella marisflavi is considered responsible for infection in juvenile sea cucumber, Apostichopus japonicas. A bacteriophage specific to S. marisflavi was isolated from seafood wholesale market sewage and designated as vB_SmaM_PSM (PSM‐1). The phage was classified as a member of the Myoviridae family by negative staining transmission electronic microscopy. The stability of phage PSM‐1 was observed after treatment with different temperature gradients and pH values. The results showed that the lytic activity of phage PSM‐1 was optimum at 28 C with a pH of 7.0 and ceased at temperatures higher than 60 C. A one‐step growth curve analysis of the phage revealed eclipse and latent periods of 20 and 40 min, respectively, with a burst size of 219 plaque forming units per infected cell. The ability of phage PSM‐1 to inhibit the growth of S. marisflavi AP629 was tested in vitro using the phage with three multiplicity of infection (MOI) values of 0.1, 1, and 10. All treated cultures that produced an inhibition of the growth of S. marisflavi AP629 were compared with the untreated culture, and the treatment group with a MOI value of 10 showed the highest inhibition effect among the three doses. In an in vivo performance experiment, the sea cucumbers (15–20 g) were randomly assigned to five groups (one negative control, one antibiotic treatment group, and three phage treatment groups) in a phage injection trial as well as a phage immersion trial. All groups were infected with S. marisflavi strain AP629 (3.3 × 106 colony forming units/g, LD50 [lethal dose 50%] level) by intraperitoneal injection. After 8 d of observation, the survival rates ranged from 40 to 80% in all phage treatment groups, while 10% in the negative control group. The optimal therapeutic time points of the two trials were also determined. The results showed that using phage prior to infection could be a good choice to protect sea cucumbers. The overall results of this experiment indicate that using phage may be a feasible biological control agent to prevent S. marisflavi infection in sea cucumbers.

Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, β-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to>512, 16, 16, and 256μg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85μmol/min/ml) and ARST(CIP) (48.83μmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95% for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52% for ARST(CIP) at 15min of infection. The least lytic activity of phage was 20% against the ARST(CIP), followed by ASST(CIP) (30%). The adsorption rate of phage against ARST(CIP) was 52% at 15min of infection, which resulted in the decrease in lytic activity (12%). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens.

Effect of Urea upon Activity of Bacteriophage.

In attempting to minimize denaturation of phage by heat we employed a number of substances which various investigators have found to prevent denaturation of proteins.1 While some of these substances delayed denaturation of phage (saccharose, glycerine and to lesser extent CaCl2 or glucose) other substances (sodium salicylate, Bayer 205, or urea) exhibited no gross protective effect. During these experiments it was noted, however, that when mixtures containing phage and urea were plated with homologous bacteria at intervals during the experiment (before the inactivation of phage has occurred) the plaques of lysis appeared to be larger and noticeably clearer than on control plates containing the phage without the addition of urea. This apparent intensification of lysis in the presence of urea suggested the possibility that addition of urea may perhaps bring out the lytic activity of phage under circumstances where lysis is ordinarily inhibited, as for instance on 4% agar. As we have shown, under these circu...